Lactic acid polymers, like for instance poly(D,L-lactide-co-glycolide)copolymers (PLGA), are biodegradable polymers and well known in the art for example from EP1468035, U.S. Pat. No. 6,706,854, WO2007/009919A2, EP1907023A, EP2263707A, EP2147036, EP0427185 or U.S. Pat. No. 5,610,266.
US 2005/0053590A1 describes an endothelium-targeting nanoparticle for reversing endothelial dysfunction. A method for ameliorating cellular dysfunction comprises the steps of providing a composition that specifically targets a dysfunctional endothelial cell comprising a targeting ligand that binds specifically to an endothelial cell and a nucleic acid and delivering the composition to the cell under conditions that increase intracellular tetrahydrobiopterin concentration. The composition may further comprise a nanoparticle selected from a long list of suitable types of nanoparticles including calcium phosphate nanoparticles and biodegradable nanoparticles formulated from poly (D,L-lactide-co-glycolide)(PLGA) or combinations of the different nanoparticle types mentioned there.
WO 2007/048599 describes particulate drug delivery systems based on a polymeric carrier, characterized in that at least one signal substance for transport through a biological barrier and at least one active ingredient are included, with carrier, signal substance and active ingredient showing no covalent linkages with one another. The signal substance (cell penetrating peptide (CPP)) is lactoferrin or a peptide derived from lactoferrin.
In a particularly preferred embodiment, a signal peptide with the amino acid sequence
KCFQWQRNMRKVRGPPVSCIKR (SEQ ID No.1 (=SEQ IDNo. 3 in WO2007/048599)), CFQWQRNMRKVRGPPVSC (SEQ ID No.2 (=SEQ ID No. 4in WO2007/048599)), FQWQRNMRKVRGPPVS (SEQ ID No.3 (=SEQ ID No. 5in WO2007/048599)), FQWQRNMRKVR (SEQ ID No.4 (=SEQ ID No. 6 inWO2007/048599)), KCRRWQWRMKKLGAPSITCVRR (SEQ ID No.5 (=SEQ ID No.29 in WO2007/048599))and CRRWQWRMKKLGAPSITC (SEQ ID No.6 (=SEQ ID No. 30 in WO2007/048599))or a derivative thereof.
In a preferred embodiment, the cell-penetrating peptides of WO 2007/048599 are comprising an amino acid sequence as shown in WO 2007/048599 in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 29 or SEQ ID No. 30 or a corresponding sequence with an identity of at least 40%, preferably of at least 50%, particularly preferably with an identity of more than 75% or better of more than 90%.
WO 2007/076904A1 describes a peptide having an amino acid sequence comprising at least 8 consecutive amino acids of the human lactoferrin protein or of the bovine lactoferrin protein, whereby the peptide is suitable to act as a cell-penetrating peptide (CPP). Many of the peptides mentioned in WO 2007/076904A1 and in WO 2007/048599 are identical.
The most promising cell-penetrating peptide with the best effects in the examples is KCFQWQRNMRKVRGPPVSCIKR (SEQ ID No. 1 (=SEQ ID No. 3 in WO 2007/048599 and in WO 2007/076904A1)).
The lactoferrin-derived cell-penetrating peptides are intended to permit the transport of cargo molecules, which are active pharmaceutical ingredients such as DNA, RNA, peptides or antigens for vaccination, which may be orally ingested, through the biological membranes and thus allow an efficient uptake of these molecules in the human or animal organism.
WO2014/141288A1 (International publication date 18 Sep. 2014) describes a nanomaterial showing multi-functional properties such as radioactivity, raman scattering, near-infrared (NIR) fluorescence, para- or superparamagnetism and X-ray absorption. The multifunctional nanocontrast agent may have spherical or non-spherical shape and size ranging from 1-200 nm and can be delivered intravenously, intramuscularly or orally. The nanomaterial is based on calcium phosphate nanoparticles. The nanoparticles functions as multifunctional nanocontrast agent that may be conjugated or loaded with drug molecules such as bisphosphonates, chemodrugs, anticancer gene therapy agents, RNA fragments (siRNA, mi-RNA), photosensitive drugs, small molecule inhibitors, antibiotics. The calcium phosphate nanoparticles may be formulated in a polymeric shell of a biodegradable polymer containing the drugs. The biodegradable polymer may be among others a poly-lactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethyleneimine (PEI), chitosan or carboxymethyl chitosan. The nanoparticles may be conjugated on their surface with targeting ligands such as including folic acid, antibodies, peptides, aptamers or carbohydrates. Capping agents such as citrate, polymers such as PEG, polyethyenimine, biphosphonates may be added.
Norihiro Watanabe et al. describe “Transgenic Expression of a Novel Immunosuppressive Signal Converter on T-cells”, Molecular Therapy, vol. 21, 2013-05-01, p. s153-S153, (XP055131645).
Ping Zeng et al.: “Chitosan-modified poly/,-lactide-co-glycolide) nanospheres for plasmid DNA delivery and HBV gene silencing”, International Journal of Pharmaceutics, Elsevier BV, NL, vol. 415, 2011-05-20, p. 259-266 (XP028099873, ISSN: 0378-5173). Ping Zeng et al describe nanoparticles formulated using poly(lactic-co-glycolic acid) (PLGA) for plasmid DNA (pDNA) delivery. Jie Tang et al. describes in Acta Pharmaceutica Sinica 2013, 48 (2): 298-304, the preparation and in vitro evaluation of calcium phosphate-pDNA nanoparticles (pDNA-CaPi) which are encapsulated in poly(lactid-co-glycolid)-copolymer (PLGA) in a core/shell (CS) structure. The core/shell structure particles (CS-pDNA-CaPi-PLGA-NPs) are compared to embedded CaPi modified PLGA nanoparticles (embedded-pDNA-CaPi-PLGA-NPs). The core/shell structure nanoparticles (CS-pDNA-CaPi-PLGA-NPs) were spherical in shape with an average particle size of 155+/−4.5 nm, zeta potentials of −0.38+/−0.1 mV, an entrapment efficiency of 80.56+/−2.5% and a loading efficiency of 1.16+/−0.04%. The core/shell structure particles were stable in the release media and could protect pDNA against nuclease degradation. They also exhibited sustained release of pDNA in vitro. The highest gene transfection efficiency of the CS-pDNA-CaPi-PLGA-NPs in vitro reached (24.66+/−0.46)% after 72 h transfection, which was significantly higher than that of free pDNA [(0.33+/−0.04)%, P<0.01] and the pDNA-PLGA-NPs [(1.5+/−0.07)%, P<0.01]. The transfection lasted for longer time than that of embedded-pDNA-CaPi-PLGA-NPs and the cytotoxicity was significantly lower than that of polyethylene-imine (PEI). Therefore CS-pDNA-CaPi-PLGA-NPs are supposed to be promising non-viral gene vectors.
Jie Tang et al: “Calcium phosphate embedded PLGA nanoparticles: A promising gene delivery vector with high gene loading and transfection efficiency”, International Journal of Phramaceutics, Elsevier BV, NL, vol. 431, 2012-04-17, p. 210-221 (XP028503199, ISSN: 0378-5173). Jie Tang et al. describes the preparation and in vitro evaluation of calcium phosphate-pDNA nanoparticles (pDNA-CaPi) which are encapsulated in poly(lactid-co-glycolid)-copolymer (PLGA). The transfection efficiency of these nanoparticles on human embryotic kidney cells was found to be much higher with pDNA loaded PLGA nanoparticles or than with CaPi-pDNA embedded PLGA microparticles.
(Mingzehn Zang et al: Nano-structured composites based on calcium phosphatefor cellular delivery of therapeutic and diagnostic agents”, Nano today, vol. 4, no. 6, 2009-12-01, p. 508-517 (XP055153407; ISSN: 1748-0132). The use of nanostructured calcium phosphate composites with emphasis on PEGylated calcium phosphate delivery systems especially for nucleic acids such as siRNA is described.